The major merozoite surface protein of Plasmodium species has been shown to be a target of varying degrees of protective immunity against the asexual blood stages in rodent and human malaria. For example, vaccination of mice with purified P230, the major merozoite surface protein of the rodent malaria Plasmodium yoelii, has resulted in reduced parasitemias in comparison to controls upon intravenous challenge with a lethal dose of parasitized erythrocytes (Holder et al. (1981) Nature 294:361). Mice have also been protected against P. yoelii by passive transfer of a monoclonal antibody (Mab) specific for P230 (Majarian et al. (1984) J. Immunol. 132:3131) and against (rodent malaria) Plasmodium chabaudi adami challenge by passive immunization with a Mab specific for the homologous 250-kDa molecule of this plasmodium species (Lew et al. (1989) Proc. Natl. Acad. Sci. USA 86:3768). The ability to confer resistance to parasite challenge by passive transfer of antibodies suggests that antibody-mediated mechanisms play an important role in antigen-specific immunity to malaria.
Despite these findings, however, no commercially viable vaccine has been developed against the major merozoite surface antigen of the major human malaria pathogen, Plasmodium falciparum. For example, using naturally derived materials, such as the precursor of the major merozoite surface protein (MSP) alone (gp195: 195,000–200,000 Da molecular species; merozoite surface protein-1 (MSP-1)), gp195 mixed with certain of its natural processing fragments, or a natural processing fragment by itself, partial protection against Plasmodium falciparum infection was achieved by some researchers (Hall et al. (1984) Nature 311:379; Perrin et al. (1984) J. Exp. Med. 160:441; Patarroyo et al. (1987) Vaccines 87 (Brown, Chanock, Lerner, ed.) Cold Spring Harbor Laboratory Press, CSH, N.Y. 117–124). An effective vaccine against Plasmodium falciparum cannot convey merely partial protection, however, since even low parasitemias of this organism can cause serious illness. A commercially useful vaccine should substantially eliminate parasitemia.
More recently, attention has been focused on the 42 kDa C-terminal processing fragment of gp-195 (p42). See Chang et al. (1996) Infect Immun 64(1):253. Co-pending U.S. patent application Ser. No. 08/195,705, the disclosure of which is incorporated by reference herein, describes a p42 peptide composition produced in a baculovirus vector and expressed in insect host cells. Similarly, co-pending U.S. patent application Ser. No. 09/500,376, the disclosure of which is incorporated by reference herein, describes a modified p42 peptide composition in combination with an adjuvant, wherein the p42 polypeptide is also produced in a baculovirus vector and expressed in insect host cells. “BVp42” as used herein refers to the expression of p42 in insect cells as described in these patents. Although capable of providing an immunogenic p42 polypeptide, the above-described methods may be cost prohibitive for commercial feasibility, particularly for a therapeutic agent primarily targeted to third world developing countries.
What is needed, therefore, is a method of producing an immunogenic p42 polypeptide which can reduce the costs associated with p42 production while still providing adequate amounts of immunogen. While transgenic plant expression is one potential option, earlier studies have demonstrated the inherent difficulties associated with expression of foreign (or non-plant) proteins in plant host cells. See, e.g., Estruch et al. (1997) Nat. Biotechnol. 15(2):137; Tian et al. (1991) Chin J Biotechnol. 7(1):1; Iannacone et al. (1997) Plant Mol. Biol. 34(3):485; and Ohme-Takagi et al. (1993) Proc Natl Acad Sci U.S.A. 90(24): 11811. Accordingly, a successful method for expressing malarial antigens in transgenic plants will have to overcome these problems.
To date, there has been only one other published study describing the expression of Plasmodium gene sequences in plants. In this study, several sequential B-cell epitopes were expressed on the surface of the Tobacco Mosaic Virus, creating plant virus particles which are transiently produced in infected tobacco tissue (Turpen et al. 1995. Biotechnology 13(1):53).